Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

Initial characterization of mitochondrial DNA control region haplotypes

of the Antillean manatee (Trichechus manatus manatus,

Sirenia:Trichechidae) in Guatemala

Grecia Mendez*1; https://orcid.org/0000-0002-8559-6557

Susan Carney2; https://orcid.org/0000-0003-3047-7435

Heidy Amelia Garcia3; https://orcid.org/0000-0002-2392-6768

Ester Quintana-Rizzo4; https://orcid.org/0000-0002-8957-0506

1. Department of Biology, Universidad del Valle, Guatemala; grecia.m.mendez@gmail.com (Correspondence*)

2. Department of Biology, Hood College, Frederick MD, USA; slc239@gmail.com

3. Fundación Defensores de la Naturaleza, Guatemala, Guatemala; hgarcia@defensores.org.gt

4. Department of Biology, Emmanuel College, Boston MA, USA; tetequintana@comcast.net

Received 21-IX-2022. Corrected 13-II-2023. Accepted 11-IV-2023.

ABSTRACT

Introduction: Small populations are at risk of losing genetic variability much faster than large populations; this

subsequently decreases their ability to adapt when facing environmental changes. A small population of the

endangered Antillean manatee (Trichechus manatus manatus) has been identified in Guatemala.

Objective: This study explored the genetic diversity of the Antillean manatee in Guatemala by analysing mito-

chondrial DNA control region haplotypes in the two most important habitats for the species, Bahía La Graciosa,

a coastal bay and Bocas del Polochic, a coastal wetland, both located in the Izabal State.

Methods: Genetic samples were collected using non or minimally invasive sampling techniques: scraping of

epidermal tissue, collection of floating feces, and collection of tissue from carcasses. DNA extractions, DNA

amplification using polymerase chain reaction (PCR), and sequencing of the control D-loop region were used to

process and analyse the samples.

Results: Seven mitochondrial DNA sequences were obtained from 36 samples collected (minimum of four and

maximum of seven individuals). Four haplotypes were identified, A01, A03, A04, and J01. No other Central

American country has reported this number of haplotypes in a manatee population, and it is the first time that

haplotype A01 has been reported for the region. The Guatemalan manatee population comprises at least two

genetic lineages, the Florida/Greater Antilles lineage (haplotypes A01, A03, and A04) and the Mesoamerican

lineage (J01).

Conclusion: Further studies, with the use of nuclear markers, are necessary to understand the population

dynamics between Bahia La Graciosa and Bocas del Polochic to identify the number of management units pres-

ent in the country; also, the degree of relatedness with the Belizean population needs to be established to better

coordinate conservation efforts.

Key words: Non-invasive genetic sampling; endangered species; control D-loop region; Lago de Izabal; Atlantic

coast of Guatemala; conservation management plans.

RESUMEN

Caracterización inicial de haplotipos de la región control de ADN mitocondrial del Manatí Antillano

(Trichechus manatus manatus Sirenia:Trichechidae) en Guatemala

Introducción: Las poblaciones pequeñas corren el riesgo de perder variabilidad genética mucho más rápido que

una población de mayor tamaño; disminuyendo, así mismo, su capacidad de adaptarse ante cambios ambientales.

https://doi.org/10.15517/rev.biol.trop..v71iS4.57278

SUPPLEMENT • MANATEES

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

INTRODUCTION

Genetic studies on the West Indian mana-

tee, Trichechus manatus Linnaeus, 1758 have

shown low haplotype and nucleotide diversity,

with 29 haplotypes identified to date, distrib-

uted in three distinct lineages: Cluster I, Cluster

II, and Cluster III (Alvarez-Aleman et al., 2022;

Caballero et al., 2021; Díaz-Ferguson et al.,

2017; Garcia-Rodriguez et al., 1998; Satizabal et

al., 2012; Vianna et al., 2006). Cluster I includes

haplotypes found in Florida and the Greater

Antilles, Cluster II is distributed from the Gulf

of Mexico to the Caribbean coast of South

America, and Cluster III is exclusive to Brazil

and Guyana (Vianna et al., 2006). The Antillean

manatee (T. m. manatus), classified as endan-

gered on the IUCN Red List (Self-Sullivan &

Mignucci-Giannoni, 2008), is one of the two

subspecies of the West Indian manatee and is

found in small population pockets throughout

most of its range.

In Central America, the genetic variability

of the Antillean manatee has been studied in

Belize (Hunter et al., 2010) and Panama (Díaz-

Ferguson et al., 2017); these countries share the

Cluster II lineage (Díaz-Ferguson et al., 2017).

Belize has the largest manatee population in the

western hemisphere with around 1 000 animals

(Hunter et al., 2010) and, through conservation

efforts, could assist in the recovery of the adja-

cent Guatemalan population (Quintana-Rizzo

& Reynolds III, 2010). The most recent estimate

is 150 individuals in Guatemala (Quintana-

Rizzo & Reynolds III, 2010), and threats like

illegal hunting limit the survival of the popu-

lation (Machuca-Coronado & Corona, 2019).

The objective of this study was to explore the

genetic diversity of the Antillean manatee in

Guatemala in two areas that are ecologically

distinct but important for the species, Bocas

del Polochic, an inshore wetland and Bahia La

Graciosa, a coastal bay (Fig. 1A).

Manatee epidermal tissue, feces, and car-

cass skin cuts were collected at the two study

sites between July 2012 and February 2011. To

obtain epidermal tissue, a modified method,

initially developed by Carney et al. (2007) was

employed. A scraper with increased perfora-

tions (from 2 mm to 5 mm wide) and a lighter

pole (4 m of aluminum instead of 2 m of PVC)

were used to scrape the dorsal part of the

manatee without hurting the animal and subse-

quently storing the tissue with 75 % ethanol and

Una pequeña población de la especie en peligro de extinción el manatí antillano (Trichechus manatus manatus)

ha sido identificada en Guatemala.

Objetivo: Este estudio explora la diversidad genética del manatí antillano en Guatemala mediante el análisis de

haplotipos de la región de control del ADN mitocondrial en los dos hábitats más importantes identificados para

la especie, Bahía La Graciosa, un bahía costera y Bocas del Polochic, un humedal costero, ambos localizados en

el departamento de Izabal.

Métodos: Las muestras genéticas se colectaron utilizando técnicas de muestreo no invasivas o mínimamente

invasivas: raspado de tejido epidérmico, recolección de heces y recolección de tejido extraído de cadáveres. Se

usaron extracciones de ADN, amplificación de ADN médiate la reacción en cadena de la polimerasa (PCR) y

secuenciación de la región control D-loop.

Resultados: Se obtuvieron un total de siete secuencias de ADN mitocondrial de 36 muestras recolectadas. Cuatro

haplotipos fueron identificados, A01, A03, A04 y J01. Ningún otro país centroamericano ha reportado esta

cantidad de haplotipos en una población de manatíes y es la primera vez que se reporta el haplotipo A01 para

la región. La población de manatíes guatemaltecos comprende al menos dos linajes genéticos, el linaje Florida/

Antillas Mayores (haplotipos A01, A03 y A04) y el linaje mesoamericano (J01).

Conclusión: Son necesarios más estudios, con el uso de marcadores nucleares, para comprender la dinámica

poblacional entre Bahía La Graciosa y Bocas del Polochic y para poder identificar el número de unidades de

manejo presentes en el país: además, se debe establecer el grado de relación con la población de Belice para coor-

dinar mejor los esfuerzos de conservación.

Palabras clave: Muestro genético no invasivo; especie en peligro de extinción; región control D-loop; Lago de

Izabal; Costa Atlántica de Guatemala; planes de manejo de conservación.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

1 X TE (pH 8). After a manatee sighting, fecal

samples were collected within a 100 m radius

of the sighting. These were collected using

new disposable plastic bags and latex gloves

(Muschett et al., 2009) and were stored at room

temperature in sterile containers with 95 %

ethanol using a proportion of 1:3 (Muschett et

al., 2009). Lastly, skin cuts from dead carcasses

were collected opportunistically and stored at

-20 °C. Two extraction protocols were used to

treat tissue samples with different degradation

states: the phenol-chloroform method (Mus-

chett et al., 2009) was used for samples with

high DNA concentration and a silica method

(Höss & Pääbo, 1993) was used for low DNA

concentrations. Two extraction protocols were

also used to assay fecal samples: Zhang et al.

(2006) and Marrero et al. (2009). In the Zhang

et al. (2006) protocol, instead of binding the

DNA to a spin column with guanidine thiocya-

nate, the samples were resuspended in 500 µl

of isopropanol at -20 °C. A modification was

used to increase the purity index of the extrac-

tions to 1.6–2.0. This modification consisted of

applying the silica method (Marrero, 2009) to

the resuspended samples.

Mitochondrial control region D-loop DNA

was amplified by PCR using 1X Buffer (Pro-

mega), 4 mM of MgCl2, 150 µM of each dNTPs,

0.3 µM of each primer (CR-4 and CR-5), 1.5 U

of Taq DNA polymerase (Promega) and 1 µl

of extracted DNA with a concentration of 100

ng/µl in a total reaction volume of 25 µl. The

PCR cycling conditions were: 94 °C for 3 min,

followed by 35 cycles of 94 °C for 1 min, 47 °C

for 1 min, 72 °C for 1 min, and a final extension

Fig. 1. (A) Location of the two sampling sites and protected areas: Bocas del Polochic Wildlife Refuge in Lago de Izabal

and Bahía La Graciosa, part of the Punta de Manabique Wildlife Refuge, both located on the Atlantic coast of Guatemala.

(B) Manatee mitochondrial control region D-loop haplotypes found in Guatemala and other parts of the Americas and the

number of samples analyzed per country. Each haplotype is depicted in distinct colors and grouped into its corresponding

cluster, previously defined by Vianna et al. (2006). Data sources: Mexico, Florida and the Dominican Republic (Vianna et

al., 2006), Cuba (Alvarez-Alemán et al., 2022), Puerto Rico (Hunter et al., 2012), Colombia (Caballero et al., 2021), Panama

(Díaz-Ferguson et al., 2017), and Belize (Hunter et al., 2010). (C) Number of genetic haplotypes (HT) and sample size (N)

for Antillean manatee in Guatemala and other Central American countries (Belize and Panama). Since floating fecal samples

cannot be assigned to different manatees, the sample size of Guatemalan manatees in this study could be less than seven,

which is the number of sequences reported but not less than four because four different haplotypes were identified.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

of 72 °C for 1 hr (García-Rodríguez et al.,

1998). The optimum annealing temperature

for fecal samples was 54 °C instead of 47 °C.

PCR products were run on a 1 % agarose gel

and visualized by electrophoresis to confirm

amplification of the expected ~ 410 bp size. The

dNTPs and primers were removed using the

Novagen purification kit. The PCR products

were bi-directionally sequenced (Macrogen

Inc., Korea) to ensure a high confidence level

in each nucleotide.

ChromasPro (2.1.10.1) was used to assem-

ble the forward and reverse strands into con-

tigs. A BLAST search of each contig was used

to identify the top match haplotype, previously

registered and defined by Vianna et al. (2006).

A Clustal Omega alignment was generated

between the contig and the haplotype, where

shared polymorphisms and percentage align-

ment were used to define each sample as its

respective haplotype.

A total of 36 manatee samples were col-

lected. Of these, 29 samples (27 fecal samples

and two tissue samples from carcasses) were

obtained in Bahía La Graciosa, and seven sam-

ples (two fecal samples and five tissue samples

from carcasses) were obtained in Bocas del

Polochic. No epidermal tissue samples were

collected during the study. Noise reduction

was a primary challenge for approaching

manatees since another research was simul-

taneously being carried out in the same boat.

This field experience limited the application of

this specific method in the region. However,

approaching manatees is delicate due to their

elusive behavior.

DNA was amplified from 22 of the samples

collected; all came from Bahía La Graciosa.

Seven samples were successfully sequenced;

one came from tissue samples and six from fecal

samples. The main reason for a lack of sequence

results from tissue samples was contamination;

the preservation, storage, and management of

samples was not optimal. On the other hand,

the limiting factor for processing fecal samples

was degradation; fecal samples degrade very

quickly when exposed to UV radiation.

Double coverage sequences ranged in

length from 390-444 bp. Two sequences had

read lengths of less than 410 bp, the length of

characterized haplotypes in Genbank. Howev-

er, these sequences of 390 and 392 bp in length

were 100 % matches to the J01 haplotype, the

most variable of the haplotype sequences iden-

tified here. To confirm each haplotype, forward

and reverse chromatograms for each manatee

sequence were aligned with the reference hap-

lotype from Genbank (Supplementary Mate-

rial 1). In all cases, there was a 100 % match

between the reference sequence and the chro-

matograms. Only one sequence can be con-

clusively traced back to an individual since it

consisted of a tissue sample from a carcass. The

remaining samples consisted of feces collected

exclusively in the bay area; therefore, there

is a risk that a single individual was sampled

multiple times and/or that these sequences

could only be representative of a few bay area

resident individuals.

Four haplotypes were identified, A01, A04,

A03 and J01 (Fig. 1B and Fig. 1C), with respec-

tive frequencies of 43 %, 14 %, 14 %, and 29 %.

These haplotypes have all been found in Cen-

tral America, except for haplotype A01 (Fig.

1B). All manatee sequences have been reg-

istered in GenBank (OQ587957-OQ587965).

Genetic diversity parameters like haplotype

frequency, haplotype diversity, and nucleotide

diversity could not be determined due to the

aforementioned risk.

This study reports four manatee mito-

chondrial control region D-loop haplotypes for

Guatemala, in a minimum of four individuals

and a maximum of seven manatees. This is

the highest number of haplotypes reported for

Mesoamerica; all other genetically studied pop-

ulations of the region, namely Belize, Panama

and the Caribbean coast of Mexico, have report-

ed three haplotypes (Díaz-Ferguson et al., 2017;

Hunter et al., 2010; Nourisson et al., 2011) (Fig.

1B). This significant finding in a relatively small

sample size strongly indicates that a more com-

prehensive population genetic analysis of the

Guatemalan manatees is warranted. Belize, the

adjacent manatee population to Guatemala, has

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

reported the haplotypes A03, A04, and J01 in

113 individuals; (Fig. 1C) (Hunter et al., 2010).

These three haplotypes have all been identified

in Guatemala, along with the A01 haplotype

that was reported in three out of seven sequenc-

es. Panama has also reported a different set of

three manatee haplotypes: J01, J02, and J03

(Díaz-Ferguson et al., 2017).

Each haplotype identified in Guatemala

was matched with the corresponding cluster

previously named and classified by Vianna et al.

(2006) (Supplementary Material 2). These clus-

ters are lineages inferred using nucleotides and

sequential divergence parameters to construct

phylogenetic relationships. Haplotypes A01,

A03, and A04 belong to Cluster I (Florida and

Greater Antilles) and haplotype J01 belongs to

Cluster II (Mesoamerica) (Vianna et al., 2006).

The presence of Cluster I and Cluster II gives

a bimodal character to the manatee popula-

tion of Guatemala. This pattern has also been

detected in manatees sampled from Mexico,

Belize, Colombia, and Venezuela (Vianna et al.,

2006), indicating that the manatees in Guate-

mala likely share a common ancestry with the

rest of the Mesoamerican manatee population.

The presence of the A01 and the A03 hap-

lotypes in the study suggests that the Guatema-

lan manatees have a genetic relationship with

the North American and/or Greater Antilles

population (Fig. 1B). Haplotype A03 has been

reported in Cuba (Alvarez-Alemán et al., 2022)

and Belize (Hunter et al., 2010). A01 is the only

haplotype reported for the Florida population

and has a broad distribution across the wider

Antilles (Hunter et al., 2012; Vianna et al.,

2006). Florida manatees are known to travel

outside their normal range [e.g., from Florida

to Cuba, Alvarez-Aleman et al. (2010); from

Florida to Mexico, Castelblanco et al. (2021)];

interestingly, the haplotype A01 has not been

reported in the Belizean manatee population,

where 16 times as many samples have been

analyzed to date (Hunter et al., 2010).

A complete mitochondrial diversity study

and an analysis of nuclear DNA markers would

provide a further understanding of the origin

and genetic status of the Guatemalan manatee

population. Nuclear DNA markers would be

fundamental in determining the number of

management units present in the country. They

would aid in delineating population genetic

relationships with neighboring countries, like

Belize, and across the region. Conservation

efforts should focus on determining the distri-

bution of each genetic lineage within the coun-

try as well as the degree of interbreeding and,

prioritize the preservation of each ancestral

lineage and promote habitat/population con-

nectivity where possible.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

See supplementary material

a04v71s4-SM1

ACKNOWLEDGMENTS

Research was conducted under research

permit 042/2010 and collection permit I-019-

2010 approved by the Guatemalan Govern-

ment, National Council for Protected Areas

(CONAP). We would like to thank our main

sources of funding, FONACON “Fondo Nacio-

nal para la Conservación de la Naturaleza”

and Defensores de la Naturaleza. We thank

the entire team of the project “Fortalecimien-

to Institucional para Consolidar el Manejo y

Conservación del Manatí (Trichechus manatus

manatus) en la Costa Atlántica de Guatemala”,

Oscar Hugo Machuca, Arnoldo Chaal, and

Marco Tulio Milla who made the fieldwork

possible. To the Department of Biology of the

University, Universidad del Valle de Guatemala,

for making the laboratory part of the study a

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57278, diciembre 2023 (Publicado Nov. 01, 2023)

success along with the careful mentorship from

Andres Avalos. We are eternally grateful for

everyone’s involvement in this project.

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